Antiviral therapy

ABSTRACT

The invention relates to a combination comprising (3S, 11aR)—N-[(2,4-difluorophenyl)methyl]-2,3,5,7,11,11a-hexahydro-6-hydroxy-3-methyl-5,7-dioxo-oxazolo [3,2-a] pyrido [1,2-d] pyrazine-8-carboxamide, or a pharmaceutically acceptable salt thereof and rilpivirine, along with therapeutic methods administering the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of Ser. No. 16/835,733 filed Mar. 31,2020, which is a continuation of Ser. No. 15/838,738 filed Dec. 12,2017, abandoned, which is a continuation of Ser. No. 15/366,566, filedDec. 1, 2016, abandoned, which is a continuation of Ser. No. 15/073,728,filed Mar. 18, 2016, abandoned, which is a continuation of Ser. No.14/710,952 filed May 13, 2015, abandoned, which is a continuation ofSer. No. 13/575,380 filed Jul. 26, 2012, abandoned, which is a NationalPhase of International Application No. PCT/US2011/022219, filed Jan. 24,2011 which claims priority from U.S. Provisional Application Ser. No.61/298,589 filed Jan. 27, 2010.

BACKGROUND OF THE INVENTION

The human immunodeficiency virus (“HIV”) is the causative agent foracquired immunodeficiency syndrome (“AIDS”), a disease characterized bythe destruction of the immune system, particularly of CD4⁺ T-cells, withattendant susceptibility to opportunistic infections, and its precursorAIDS-related complex (“ARC”), a syndrome characterized by symptoms suchas persistent generalized lymphadenopathy, fever and weight loss. HIV isa retrovirus; the conversion of its RNA to DNA is accomplished throughthe action of the enzyme reverse transcriptase. Compounds that inhibitthe function of reverse transcriptase inhibit replication of HIV ininfected cells. Such compounds are useful in the prevention or treatmentof HIV infection in humans.

In addition to CD4, HIV requires a co-receptor for entry into targetcells. The chemokine receptors function together with CD4 asco-receptors for HIV. The chemokine receptors CXCR4 and CCR5 have beenidentified as the main co-receptors for HIV-1. CCR5 acts as a majorco-receptor for fusion and entry of macrophage-tropic HIV into hostcells. These chemokine receptors are thought to play an essential rolein the establishment and dissemination of an HIV infection. Therefore,CCR5 antagonists are thought to be useful as therapeutic agents activeagainst HIV.

As in the case of several other retroviruses, HIV encodes the productionof a protease which carries out post-translational cleavage of precursorpolypeptides in a process necessary for the formation of infectiousvirions. These gene products include pol, which encodes the virionRNA-dependent DNA polymerase (reverse transcriptase), an endonuclease,HIV protease, and gag, which encodes the core-proteins of the virion.

One focus of anti-viral drug design has been to create compounds whichinhibit the formation of infectious virions by interfering with theprocessing of viral polyprotein precursors. Processing of theseprecursor proteins requires the action of virus-encoded proteases whichare essential for replication. The anti-viral potential of HIV proteaseinhibition has been demonstrated using peptidyl inhibitors.

A required step in HIV replication in human T-cells is the insertion byvirally-encoded integrase of proviral DNA into the host cell genome.Integration is believed to be mediated by integrase in a processinvolving assembly of a stable nucleoprotein complex with viral DNAsequences, cleavage of two nucleotides from the 3′ termini of the linearproviral DNA and covalent joining of the recessed 3′ OH termini of theproviral DNA at a staggered cut made at the host target site. The repairsynthesis of the resultant gap may be accomplished by cellular enzymes.Inhibitors of HIV integrase can be effective in treating AIDS andinhibiting viral replication.

Administration of combinations of therapeutic compounds in the treatmentof HIV infection and related conditions can result in potentiatedantiviral activity, reduced toxicity, delayed progression to resistance,and increased drug efficacy. Combinations administered in a singledosage unit can result in increased patient compliance as the pillburden is reduced and dosing schedules are simplified. However, not allcompounds are suitable for administration in combinations. Factors thatinfluence the feasibility of combinations include the chemicalinstability of the compounds, size of the dosage unit, potential forantagonistic or merely additive activities of the combined compounds,and difficulties in achieving a suitable formulation.

There is continued need to find therapeutic agents suitable for use incombination and feasible pharmaceutical compositions to treat HIVinfection. Due to their high potency and pharmacokinetic profile,certain HIV integrase inhibitors are attractive as components incombination therapy.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Inhibition of HIV-1_(IIIB) by a compound of formula (I),GSK1349572A, in combination with abacavir (ABC).

FIG. 2: Inhibition of HIV-1_(IIIB) by a compound of formula (I),GSK1349572A, in combination with efavirenz (EFV).

FIG. 3: Inhibition of HIV-1_(IIIB) by a compound of formula (I),GSK1349572A, in combination with lopinavir (LPV)

SUMMARY OF THE INVENTION

The present invention relates to combinations of compounds comprisingHIV integrase inhibitors and other therapeutic agents. Such combinationsare useful in the inhibition of HIV replication, the prevention and/ortreatment of infection by HIV, and in the treatment of AIDS and/or ARC.The present invention also features pharmaceutical compositionscontaining HIV integrase inhibitors.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to combinations comprising a compound ofthe following formula (I), (II), or (III):

or a pharmaceutically acceptable salt thereof, and one or moretherapeutic agents selected from the group consisting of nucleotidereverse transcriptase inhibitors, nucleoside reverse transcriptaseinhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors(NNRTIs), protease inhibitors, CCR5 antagonists, CXCR4 antagonists,fusion inhibitors, maturation inhibitors, and integrase inhibitors.

The present invention relates to methods of treatment of HIV infection,AIDS, and AIDS related conditions by administering to a subject acompound of formula (I), (II), or (III) and one or more therapeuticagents selected from the group consisting of nucleotide reversetranscriptase inhibitors, nucleoside reverse transcriptase inhibitors(NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs),protease inhibitors, CCR5 antagonists, CXCR4 antagonists, fusioninhibitors, maturation inhibitors, and integrase inhibitors.

A compound of formula (I) is also known as GSK1349572. A chemical nameof the compound of formula (I) is (4R,12aS)—N-[2,4-flurophenyl)methyl]-3,4,6,8,12,12a-hexahydro-7-hydroxy-4-methyl-6,8-dioxo-2H-pyrido[1′,2′:4,5]pyrazino [2,1-b] [1,3] oxazine-9-carboxamide.

A chemical name of the compound of formula (II) is (3S,11aR)—N-[(2,4-difluorophenyl)methyl]-2,3,5,7,11,11a-hexahydro-6-hydroxy-3-methyl-5,7-dioxo-oxazolo[3,2-a] pyrido [1,2-d] pyrazine-8-carboxamide.

A chemical name of the compound of formula (III) is(4aS,13aR)—N-[2,4-difluorophenyl)methyl]-10-hydroxy-9,11-dioxo-2,3,4a,5,9,11,13,13a-octahydro-1H-pyrido[1,2-a]pyrrolo[1′,2′:3,4,]imidazo[1,2-d]pyrazine-8-carboxamide.

The term “pharmaceutically acceptable carrier or adjuvant” refers to acarrier or adjuvant that may be administered to a patient, together witha compound of this invention, and which does not destroy thepharmacological activity thereof and is nontoxic when administered indoses sufficient to deliver a therapeutic amount of the antiviral agent.

The term “treatment” as used herein refers to the alleviation ofsymptoms of a particular disorder in a patient, or the improvement of anascertainable measurement associated with a particular disorder, and mayinclude the suppression of symptom recurrence in an asymptomatic patientsuch as a patient in whom a viral infection has become latent. Treatmentmay include prophylaxis which refers to preventing a disease orcondition or preventing the occurrence of symptoms of such a disease orcondition, in a patient. As used herein, the term “patient” refers to amammal, including a human.

As used herein, the term “subject” refers to a patient, animal or abiological sample.

Pharmaceutically acceptable salts of the compounds according to theinvention include those derived from pharmaceutically acceptableinorganic and organic acids and bases. Examples of suitable acidsinclude hydrochloric, hydrobromic, sulfuric, nitric, perchloric,fumaric, maleic, phosphoric, glycollic, lactic, salicyclic, succinic,toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic,ethanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic andbenzenesulfonic acids. Other acids, such as oxalic, while not inthemselves pharmaceutically acceptable, may be employed in thepreparation of salts useful as intermediates in obtaining the compoundsof the invention and their pharmaceutically acceptable acid additionsalts. Salts derived from appropriate bases include alkali metal (e.g.sodium), alkaline earth metal (e.g., magnesium), ammonium, NW₄ ⁺(wherein W is C1-4 alkyl) and other amine salts. An advantageous salt issodium salt.

Salts of the compounds of the present invention may be made by methodsknown to a person skilled in the art. For example, treatment of acompound of the present invention with an appropriate base or acid in anappropriate solvent can yield the corresponding salt.

The present invention relates to methods of treating or preventing viralinfection, for example an HIV infection, in a human comprisingadministering to the human a therapeutically effective amount of acompound of formula (I), (II), or (III) or a pharmaceutically acceptablesalt thereof in combination with one or more therapeutic agents selectedfrom the group consisting of nucleotide reverse transcriptaseinhibitors, nucleoside reverse transcriptase inhibitors (NRTIs),non-nucleoside reverse transcriptase inhibitors (NNRTIs), proteaseinhibitors, CCR5 antagonists, CXCR4 antagonists, fusion inhibitors,maturation inhibitors, and integrase inhibitors. The combination may beadministered simultaneously or sequentially.

The compounds of formula (I), (II) and (III) are particularly suited tothe treatment or prophylaxis of HIV infections and associatedconditions. Reference herein to treatment may extend to prophylaxis aswell as the treatment of established infections, symptoms, andassociated clinical conditions such as AIDS related complex (ARC),Kaposi's sarcoma, and AIDS dementia.

Combination therapies comprise the administration of a compound of thepresent invention or a pharmaceutically acceptable salt thereof andanother pharmaceutically active agent. The active ingredient(s) andpharmaceutically active agents may be administered simultaneously (i.e.,concurrently) in either the same or different pharmaceuticalcompositions or sequentially in any order. The amounts of the activeingredient(s) and pharmaceutically active agent(s) and the relativetimings of administration will be selected in order to achieve thedesired combined therapeutic effect.

Examples of such therapeutic agents include, but are not limited to,agents that are effective for the treatment of viral infections orassociated conditions. Among these agents are nucleotide reversetranscriptase inhibitors, acyclic nucleoside phosphonates, for example(S)-1-(3-hydroxy-2-phosphonyl-methoxypropyl) cytosine (HPMPC),[[[2-(6-amino-9H-purin-9-yl)ethoxy]methyl]phosphinylidene]bis(oxymethylene)-2,2-dimethyl propanoic acid(bis-POM PMEA, adefovir dipivoxil), adefovir,[[(1R)-2-(6-amino-9H-purin-9-yl)-1-methylethoxy]methyl] phosphonic acid(tenofovir), tenofovir disoproxil fumarate, and(R)-[[2-(6-Amino-9H-purin-9-yl)-1-methylethoxy]methyl]phosphonic acidbis-(isopropoxycarbonyloxymethyl)ester (bis-POC-PMPA); nucleosidereverse transcriptase inhibitors, for example 3′-azido-3′-deoxythymidine(AZT, zidovudine), 2′,3′-dideoxycytidine (ddC, zalcitabine),2′,3′-dideoxyadenosine, 2′,3′-dideoxyinosine (ddl, didanosine),2′,3′-didehydrothymidine (d4T, stavudine),(−)-cis-1-(2-hydroxymethyl)-1,3-oxathiolane 5-yl)-cytosine (lamivudine),cis-1-(2-(hydroxymethyl)-1,3-oxathiolan-5-yl)-5-fluorocytosine (FTC,emtricitabine),(−)-cis-4-[2-amino-6-(cyclo-propylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol(abacavir), fozivudine tidoxil, alovudine, amdoxovir, elvucitabine,apricitabine, and festinavir (OBP-601); protease inhibitors, for exampleindinavir, ritonavir, nelfinavir, amprenavir, saquinavir, fosamprenavir,lopinavir, atazanavir, tipranavir, darunavir, brecanavir, palinavir,lasinavir, TMC-310911, DG-17, PPL-100, and SPI-256; non-nucleosidereverse transcriptase inhibitors (NNRTIs), for example nevirapine,delavirdine, efavirenz, GSK2248761 (IDX-12899), lersivirine(UK-453,061), rilpivirine (TMC-278), etravirine, loviride, immunocal,oltipraz, capravirine, and RDEA-806; integrase inhibitors, for exampleraltegravir, elvitegravir, and JTK-656; CCR5 and/or CXCR4 antagonists,for example, maraviroc, vicriviroc (Sch-D), TBR-652 (TAK-779), TAK-449,PRO-140, GSK706769, and SCH-532706; fusion inhibitors, for exampleenfuvirtide (T-20), T-1249, PRO-542, ibalizumab (TNX-355), BMS-378806(BMS-806), BMS-488043, KD-247, 5-Helix inhibitors, and HIV attachmentinhibitors; and maturation inhibitors, for example, bevirimat (PA-344and PA-457).

The present invention features a combination comprising a compound offormula (I)

or a pharmaceutically acceptable salt thereof, and one or moretherapeutic agents selected from the group consisting of lamivudine,abacavir, tenofovir, efavirenz, GSK2248761, lersivirine, lopinavir,fosamprenavir, and atazanavir.

The present invention also features a combination comprising a compoundof formula (I) or a pharmaceutically acceptable salt thereof, and one ormore therapeutic agents selected from abacavir, efavirenz, or lopinavir.The present invention features a combination comprising of a compound offormula (I) or a pharmaceutically acceptable salt thereof, and abacavir.

The present invention features a method of treatment of HIV infectioncomprising administering to a subject, a compound of formula (I), or apharmaceutically acceptable salt thereof, and one or more therapeuticagents selected from the group consisting of lamivudine, abacavir,tenofovir, efavirenz, GSK2248761, lersivirine, lopinavir, fosamprenavir,and atazanavir.

The present invention features a method of treatment of HIV infectioncomprising administering to a subject, a compound of formula (I) or apharmaceutically acceptable salt thereof, with one or more therapeuticagents selected from the group consisting of abacavir, efavirenz, andlopinavir. The present invention features a method of treatment of HIVinfection comprising administering to a subject a compound of formula(I) or a pharmaceutically acceptable salt thereof, and abacavir.

The present invention features a pharmaceutical composition comprising acompound of formula (I) or a pharmaceutically acceptable salt thereof,and one or more therapeutic agents selected from the group consistingof: lamivudine, abacavir, efavirenz, tenofovir, GSK2248761, lersivirine,lopinavir, fosamprenavir, and atazanavir together with apharmaceutically acceptable carrier therefor.

The present invention features a pharmaceutical composition comprising acompound of formula (I) or a pharmaceutically acceptable salt thereof,and one or more therapeutic agents selected from the group consistingof: abacavir, efavirenz, and lopinavir, together with a pharmaceuticallyacceptable carrier therefor. The present invention features apharmaceutical composition comprising a compound of formula (I) or apharmaceutically acceptable salt thereof, and abacavir together with apharmaceutically acceptable carrier therefor.

The present invention features a combination comprising a compound offormula (II)

or a pharmaceutically acceptable salt thereof, and one or moretherapeutic agents selected from the group consisting of lamivudine,abacavir, tenofovir, efavirenz, GSK2248761, lersivirine, lopinavir,fosamprenavir, and atazanavir

The present invention also features a combination comprising a compoundof formula (II) or a pharmaceutically acceptable salt thereof, and oneor more therapeutic agents selected from abacavir, efavirenz, andlopinavir. The present invention features a combination comprising of acompound of formula (II) or a pharmaceutically acceptable salt thereof,and abacavir.

The present invention features a method of treatment of HIV infectioncomprising administering to a subject, a compound of formula (II) or apharmaceutically acceptable salt thereof, and one or more therapeuticagents selected from the group consisting of lamivudine, abacavir,tenofovir, efavirenz, GSK2248761, lersivirine, lopinavir, fosamprenavir,and atazanavir.

The present invention features a method of treatment of HIV infectioncomprising administering to a subject, a compound of formula (II) or apharmaceutically acceptable salt thereof, with one or more therapeuticagents selected from the group consisting of abacavir, efavirenz, andlopinavir. The present invention features a method of treatment of HIVinfection comprising administering to a subject a compound of formula(II) or a pharmaceutically acceptable salt thereof, and abacavir.

The present invention features a pharmaceutical composition comprising acompound of formula (II) or a pharmaceutically acceptable salt thereof,and one or more therapeutic agents selected from the group consistingof: lamivudine, abacavir, tenofovir, efavirenz, GSK2248761, lersivirine,lopinavir, fosamprenavir, and atazanavir together with apharmaceutically acceptable carrier therefor.

The present invention features a pharmaceutical composition comprising acompound of formula (II) or a pharmaceutically acceptable salt thereof,and one or more therapeutic agents selected from the group consistingof: abacavir, efavirenz, and lopinavir, together with a pharmaceuticallyacceptable carrier therefor. The present invention features apharmaceutical composition comprising a compound of formula (II) or apharmaceutically acceptable salt thereof, and abacavir together with apharmaceutically acceptable carrier therefor.

The present invention features a combination comprising a compound offormula (III)

or a pharmaceutically acceptable salt thereof, and one or moretherapeutic agents selected from the group consisting of lamivudine,abacavir, tenofovir, efavirenz, GSK2248761, lersivirine, lopinavir,fosamprenavir, and atazanavir

The present invention also features a combination comprising a compoundof formula (III) or a pharmaceutically acceptable salt thereof, and oneor more therapeutic agents selected from abacavir, efavirenz, andlopinavir. The present invention also features a combination comprisinga compound of formula (III) or a pharmaceutically acceptable saltthereof, and abacavir.

The present invention features a method of treatment of HIV infectioncomprising administering to a subject, a compound of formula (III) or apharmaceutically acceptable salt thereof, and one or more therapeuticagents selected from the group consisting of lamivudine, abacavir,tenofovir, efavirenz, GSK2248761, lersivirine, lopinavir, fosamprenavir,and atazanavir.

The present invention features a method of treatment of HIV infectioncomprising administering to a subject, a combination of a compound offormula (III) or a pharmaceutically acceptable salt thereof, with one ormore therapeutic agents selected from the group consisting of abacavir,efavirenz, and lopinavir. The present invention features a method oftreatment of HIV infection comprising administering to a subject acompound of formula (III) or a pharmaceutically acceptable salt thereof,and abacavir.

The present invention features a pharmaceutical composition comprising acompound of formula (III) or a pharmaceutically acceptable salt thereof,and one or more therapeutic agents selected from the group consistingof: lamivudine, abacavir, tenofovir, efavirenz, GSK2248761, lersivirine,lopinavir, fosamprenavir, and atazanavir together with apharmaceutically acceptable carrier therefor.

The present invention features a pharmaceutical composition comprising acompound of formula (III) or a pharmaceutically acceptable salt thereof,and one or more therapeutic agents selected from the group consistingof: abacavir, efavirenz, and lopinavir, together with a pharmaceuticallyacceptable carrier therefor. The present invention features apharmaceutical composition comprising a compound of formula (III) or apharmaceutically acceptable salt thereof, and abacavir together with apharmaceutically acceptable carrier therefor.

The present invention features combinations, methods of treatment, andpharmaceutical compositions as described above wherein apharmaceutically acceptable salt of a compound of formula (I), (II) or(III) is a sodium salt.

The present invention features combinations, methods of treatment, andpharmaceutical compositions as described above wherein one or moretherapeutic agents are a pharmaceutically acceptable salt of saidtherapeutic agents, for example, abacavir hemisulfate, fosamprenavircalcium, atazanavir sulfate, tenofovir disoproxil sulfate, vicrivirocmaleate or bevirimat dimeglumine.

The present invention features methods of treatment as described abovewherein the subject is a human.

The present invention features combinations, methods of treatment andpharmaceutical compositions as described above wherein the combinationis administered sequentially.

The present invention features combinations, methods of treatment andpharmaceutical compositions as described above wherein the combinationis administered simultaneously or concurrently.

Compounds of formula (I), (II), and (III) may be made by methodsdisclosed in WO 2006/116764, U.S. 61/193,634 (WO2010/068253) or61/193,636 (WO2010/068262), incorporated herein by reference hereto.

Abacavir may be made by methods disclosed in U.S. Pat. No. 5,034,394;5,089,500; 6,294,540; 5,641,889; 5,840,990; 5,919,941; 5,808,147;6,392,085; 6,448,403; 5,917,041; 6,087,501; 5,917,042; 6,555,687;6,552,193; 6,870,053; 6,294,540; 6,340,587; or 6,646,125.

Lamivudine may be made by methods disclosed in U.S. Pat. No. 5,047,407;7,119,202; 5,905,082; 5,696,254; 5,663,320; 5,693,787; 6,051,709; or6,329,522.

Tenofovir may be made by U.S. Pat. Nos. 5,922,695; 5,935,946; 5,977,089;6,043,230, 6,069,249.

Efavirenz may be made by may be made by methods disclosed in U.S. Pat.No. 5,519,021; 5,663,169; 5,811,423; 6,555,133; 6,639,071; or 6,939,964.

GSK2248761 may be made by methods disclosed in U.S. Pat. No. 7,534,809.

Lersivirine may be made by methods disclosed in U.S. Pat. No. 7,109,228.

Lopinavir may be made by methods disclosed in U.S. Pat. No. 5,914,332.

Fosamprenavir may be made by methods disclosed in U.S. Pat. No.6,436,989; 6,514,953; or 6,281,367.

Atazanavir may be made by methods disclosed in U.S. Pat. No. 5,849,911or 6,087,383.

The therapeutic agents of the combinations may be made according topublished methods or by any method known to those skilled in the art.

In an aspect of the invention, a compound of formula (I), (II) or (III)or a pharmaceutically acceptable salt thereof may be formulated intocompositions together with one or more therapeutic agents. Thecomposition may be pharmaceutical composition, which comprises acompound of formula (I), (II), or (III), one or more therapeutic agents,and a pharmaceutically acceptable carrier, adjuvant or vehicle. In oneembodiment, the composition comprises an amount of a combination of thepresent invention effective to treat or prevent viral infection, forexample an HIV infection, in a biological sample or in a patient. Inanother embodiment, combinations of the invention and pharmaceuticalcompositions thereof, comprising an amount of a combination of thepresent invention effective to inhibit viral replication or to treat orprevent a viral infection or disease or disorder, for example an HIVinfection, and a pharmaceutically acceptable carrier, adjuvant orvehicle, may be formulated for administration to a patient, for example,for oral administration.

The present invention features combinations according to the inventionfor use in medical therapy, for example for the treatment or prophylaxisof a viral infection, for example an HIV infection and associatedconditions. The compounds according to the invention are especiallyuseful for the treatment of AIDS and related clinical conditions such asAIDS related complex (ARC), progressive generalized lymphadenopathy(PGL), Kaposi's sarcoma, thromobocytopenic purpura, AIDS-relatedneurological conditions such as AIDS dementia complex, multiplesclerosis or tropical paraperesis, anti-HIV antibody-positive andHIV-positive conditions, including such conditions in asymptomaticpatients.

According to another aspect, the present invention provides a method forthe treatment or prevention of the symptoms or effects of a viralinfection in an infected patient, for example, a mammal including ahuman, which comprises administering to said patient a pharmaceuticallyeffective amount of a combination according to the invention. Accordingto one aspect of the invention, the viral infection is a retroviralinfection, in particular an HIV infection.

The present invention further includes the use of a combinationaccording to the invention in the manufacture of a medicament forsimultaneous (concurrent) or sequential administration to a subject forthe treatment of a viral infection, in particular and HIV infection.

The present invention further provides a method for the treatment of aclinical condition in a patient, for example, a mammal including a humanwhich clinical condition includes those which have been discussedhereinbefore, which comprises treating said patient with apharmaceutically effective amount of a compound according to theinvention. The present invention also includes a method for thetreatment or prophylaxis of any of the aforementioned diseases orconditions.

Compounds of the present invention may be administered with an agentknown to inhibit or reduce the metabolism of compounds, for exampleritonavir. Accordingly, the present invention features a method for thetreatment or prophylaxis of a disease as hereinbefore described byadministration of a compound of the present invention in combinationwith a metabolic inhibitor. Such combination may be administeredsimultaneously or sequentially.

In general a suitable dose for each of the above-mentioned conditionswill be in the range of 0.01 to 250 mg per kilogram body weight of therecipient (e.g. a human) per day, in the range of 0.1 to 100 mg perkilogram body weight per day; in the range 1 to 30 mg per kilogram bodyweight per day; in the range 0.5 to 20 mg per kilogram body weight perday. Unless otherwise indicated, all weights of active ingredients arecalculated as the parent compound of formula (I), (II), or (III) andother therapeutic agents. For salts thereof, the weights would beincreased proportionally. The desired dose may be presented as one, two,three, four, five, six or more sub-doses administered at appropriateintervals throughout the day. In some cases the desired dose may begiven on alternative days. These sub-doses may be administered in unitdosage forms, for example, containing 1 to 2000 mg; 5 to 500 mg; 10 to400 mg, 20 to 300 mg of each active ingredient per unit dosage form.

The combinations may be administered to achieve peak plasmaconcentrations of each active ingredient.

While it is possible for the active ingredients to be administeredalone, it is preferable to present it as a pharmaceutical composition.The compositions of the present invention comprise an active ingredient,as defined above, together with one or more acceptable carriers thereofand one or more additional therapeutic agents. Each carrier must beacceptable in the sense of being compatible with the other ingredientsof the composition and not injurious to the patient.

Pharmaceutical compositions include those suitable for oral, rectal,nasal, topical (including transdermal, buccal and sublingual), vaginalor parenteral (including subcutaneous, intramuscular, intravenous,intradermal, and intravitreal) administration. The compositions mayconveniently be presented in unit dosage form and may be prepared by anymethods well known in the art of pharmacy. Such methods represent afurther feature of the present invention and include the step ofbringing into association the active ingredients with the carrier, whichconstitutes one or more accessory ingredients. In general, thecompositions are prepared by uniformly and intimately bringing intoassociation the active ingredients with liquid carriers or finelydivided solid carriers or both, and then if necessary shaping theproduct.

The present invention further includes a pharmaceutical composition ashereinbefore defined wherein a compound of the present invention or apharmaceutically acceptable derivative thereof and another therapeuticagent are presented separately from one another as a kit of parts.

Compositions suitable for transdermal administration may be presented asdiscrete patches adapted to remain in intimate contact with theepidermis of the recipient for a prolonged period of time. Such patchessuitably contain the active compound 1) in an optionally buffered,aqueous solution or 2) dissolved and/or dispersed in an adhesive or 3)dispersed in a polymer. A suitable concentration of the active compoundis about 1% to 25%, preferably about 3% to 15%. As one particularpossibility, the active compound may be delivered from the patch byelectrotransport or iontophoresis as generally described inPharmaceutical Research 3(6), 318 (1986).

Pharmaceutical compositions of the present invention suitable for oraladministration may be presented as discrete units such as capsules,caplets, cachets or tablets each containing a predetermined amount ofthe active ingredients; as a powder or granules; as a solution or asuspension in an aqueous or non-aqueous liquid; or as an oil-in-waterliquid emulsion or a water-in-oil liquid emulsion. The active ingredientmay also be presented as a bolus, electuary or paste.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredients in afree-flowing form such as a powder or granules, optionally mixed with abinder (e.g. povidone, gelatin, hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (e.g. sodium starchglycollate, cross-linked povidone, cross-linked sodium carboxymethylcellulose) surface-active or dispersing agent. Molded tablets may bemade by molding a mixture of the powdered compound moistened with aninert liquid diluent in a suitable machine. The tablets may optionallybe coated or scored and may be formulated so as to provide slow orcontrolled release of the active ingredients therein using, for example,hydroxypropylmethyl cellulose in varying proportions to provide thedesired release profile. Tablets may optionally be provided with anenteric coating, to provide release in parts of the gut other than thestomach.

Pharmaceutical compositions suitable for topical administration in themouth include lozenges comprising the active ingredients in a flavoredbase, usually sucrose and acacia or tragacanth; pastilles comprising theactive ingredient in an inert basis such as gelatin and glycerin, orsucrose and acacia; and mouthwashes comprising the active ingredient ina suitable liquid carrier.

Pharmaceutical compositions suitable for vaginal administration may bepresented as pessaries, tampons, creams, gels, pastes, foams or spray.Pharmaceutical compositions may contain in addition to the activeingredient such carriers as are known in the art to be appropriate.

Pharmaceutical compositions for rectal administration may be presentedas a suppository with a suitable carrier comprising, for example, cocoabutter or a salicylate or other materials commonly used in the art. Thesuppositories may be conveniently formed by admixture of the activecombination with the softened or melted carrier(s) followed by chillingand shaping in molds.

Pharmaceutical compositions suitable for parenteral administrationinclude aqueous and nonaqueous isotonic sterile injection solutionswhich may contain anti-oxidants, buffers, bacteriostats and soluteswhich render the pharmaceutical composition isotonic with the blood ofthe intended recipient; and aqueous and non-aqueous sterile suspensionswhich may include suspending agents and thickening agents; and liposomesor other microparticulate systems which are designed to target thecompound to blood components or one or more organs. The pharmaceuticalcompositions may be presented in unit-dose or multi-dose sealedcontainers, for example, ampoules and vials, and may be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example water for injection, immediatelyprior to use. Extemporaneous injection solutions and suspensions may beprepared from sterile powders, granules and tablets of the kindpreviously described.

Unit dosage pharmaceutical compositions include those containing a dailydose or daily subdose of the active ingredients, as hereinbeforerecited, or an appropriate fraction thereof.

Pharmaceutical compositions of the present invention may be presented aspatient packs containing one or more courses of treatment in a singlepackage, for example, a blister pack. It will be understood that theadministration of the combination of the invention by means of a singlepatient pack, or patient packs of each composition, is an additionalfeature of the invention.

It should be understood that in addition to the ingredients particularlymentioned above the pharmaceutical compositions of this invention mayinclude other agents conventional in the art having regard to the typeof pharmaceutical composition in question, for example, those suitablefor oral administration may include such further agents as sweeteners,thickeners and flavoring agents.

EXAMPLES Example 1: Biological Activity Assays Method

Antiviral HIV activity was measured by means of a tetrazolium-basedcolorimetric procedure in the human T-cell leukemia virus (HTLV-1)transformed cell line MT-4. Aliquots of test compound were dilutedvertically across a deep-well master assay plate, in medium (RPMI 1640,10% vol./vol. fetal bovine serum (FBS), and 10 μg/mL gentamicin), atconcentrations that were approximately 40-fold higher than the finalassay concentration. Serial dilutions were made at either 1:2 or 1:3.16ratios. HIV inhibitors were diluted horizontally across master assayplates, also in concentrations that were approximately 40-fold higherthan the final assay concentration. Small aliquots of both thevertically-diluted and the horizontally-diluted compounds were combinedin daughter plates using an automated 96-well pipetting system(RapidPlate-96, Zymark Corp.). Checkerboard style dilutions werearranged so that every concentration of test compound was tested in thepresence and absence of every concentration of the HIV inhibitors.Anti-HIV activity tests were performed in triplicate assays, or more, ofeach combination. Exponentially growing MT-4 cells were harvested andcentrifuged at 1,000 rpm for 10 minutes in a Jouan centrifuge (Model CR4 12). Cell pellets were re-suspended in fresh medium (RPMI 1640, 20%vol./vol. FBS, 20% vol./vol. IL-2, and 10 μg/mL gentamicin) to a densityof 1.25×10⁶ cells/mL. Cell aliquots were infected by the addition ofHIV-1 (strain IIIB) diluted to give a viral multiplicity of infection(MOI) of 73 pfU per 1×10⁴ cells. A similar cell aliquot was diluted withmedium to provide a mock-infected control. Cell infection was allowed toproceed for 1 hour at 37° C. in a tissue culture incubator withhumidified 5% CO₂ atmosphere. After the 1 hour incubation the virus/cellsuspension was added to each well of the plates containing pre-dilutedcompounds. Plates were then placed in a tissue culture incubator withhumidified 5% CO₂ for 5 days. At the end of the incubation period, 40 μLof CellTiter 96 MTS reagent (Promega no. G3581) was added to each wellof the incubation plate. Plates were incubated at 37° C. for 2 to 3hours to allow for color development. O.D. was measured at 492 nM usinga microplate absorbance reader (Tecan no. 20-300).

Virus Used

HIV-1 strain IIIB, wild-type laboratory strain, virus titer=6.896 E4TCID₅₀/mL.

Data Analysis

Although some assay formats might theoretically miss antagonism due tocombination cytotoxicity, the approach described here should not miss anantagonistic effect. The readout in the MT-4 cell assay utilizes MTS, atetrazolium-based staining reagent where changes in optical density(O.D.) of the reagent are used to estimate the total cell numberremaining after treatment. Final MT-4 cell numbers may decrease due totwo effects. First, an HIV-induced cytotoxicity may occur when HIV killsgreater than 75% of the MT-4 cells during the 5 days followinginfection. Second, a compound-induced cytotoxicity may occur, where thecompound either directly kills the MT-4 cells or prevents cell growth(stasis) over the 5 days in either infected or uninfected cells. Ineither of these situations the O.D. is low as compared with infectedcells protected by anti-HIV-1 compounds or relative to untreated anduninfected control cells. Since both cytotoxic effects and antagonism ofanti-HIV activity would lead to lower O.D. we should not miss anantagonistic effect due to combination cytotoxicity, but couldunderestimate synergistic combinations.

Within assay combination cytotoxicity was evaluated by comparing wellscontaining the uninfected MT-4 cells from the assay plates thatcontained the highest concentration of test compound or the comparatorcompound, with wells containing HIV-1 infected MT-4 cells under thecorresponding highest combination concentrations. For each of thesevalues there is one well per assay plate and thus at least 3 wells percombination assay. Although they do not comprise a formal combinationcytotoxicity analysis, the ratio of compound in combinations to compoundalone provides a measure of the compound combination cytotoxicity withinthe concentrations examined.

The interaction of each pair of compound combinations was analyzed bythe methods described by Selleseth, D. W. et al. (2003) AntimicrobialAgents and Chemotherapy 47:1468-71. Synergy and antagonism are definedas deviations from dosewise additivity, which results when two drugsinteract as if they were the same drug. Values for average deviationfrom additivity in the range of −0.1 to −0.2 indicate weak synergy andvalues that approach −0.5 would indicate strong synergy of theinteraction. Conversely, positive values of 0.1 to 0.2 would indicatethat a weak antagonism exists between the treatments.

Results

A compound of formula (I) was found to be additive with raltegravir,adefovir, and maraviroc and was not affected by the presence ofribavirin. A compound of formula (I) was found to be synergistic withstavudine, abacavir, efavirenz, nevirapine, lopinavir, amprenavir,enfuvirtide.

What is claimed is:
 1. A combination comprising (3S,11aR)—N-[(2,4-difluorophenyl)methyl]-2,3,5,7,11,11a-hexahydro-6-hydroxy-3-methyl-5,7-dioxo-oxazolo[3,2-a] pyrido [1,2-d]pyrazine-8-carboxamide, or a pharmaceuticallyacceptable salt thereof and rilpivirine.
 2. A method of treatment of HIVinfection comprising administering to a human (3S,11aR)—N-[(2,4-difluorophenyl)methyl]-2,3,5,7,11,11a-hexahydro-6-hydroxy-3-methyl-5,7-dioxo-oxazolo[3,2-a] pyrido [1,2-d] pyrazine-8-carboxamide, or a pharmaceuticallyacceptable salt thereof, with rilpivirine.
 3. The method according toclaim 2, wherein the compounds are administered simultaneously.
 4. Themethod according to claim 2, wherein the compounds are administeredsequentially.